The long-term goal of the proposed work is to elucidate the cellular and molecular mechanisms that control neural crest cell diversification and differentiation. The application focuses on the differentiation of neural crest stem cells into small diameter sensory neuroblasts. It is based on prior data which indicate 3 additional roles of SCF in addition to the known trophic action in pigment cell formation: (1) SCF is a survival factor for neural crest stem cells, (2) SCF is involved in the expression of the neurotrophin receptors, p75NTR and TrkC, and (3) SCF promotes differentiation of the stem cell into small diameter sensory neuroblasts. In aim 1 the role of the SCF receptor, c-kit, is investigated by knock-down and rescue of quail c-kit, and by analyzing the mouse mutants Dominant spotting (W; c-kit defect) and Steel (Sl; SCF defect) for neural crest-related deficiencies. It is hypothesized that SCF promotes expression of p75NTR with higher probability than TrkC, thus creating at least two subsets of cells (p75NTR+/TrkC+, p75NTR+/TrkC-), which possibly have restricted developmental potentials. This issue will be addressed by in vitro clonal analysis of immunoselected cells. Aim 2 is concerned with neurotrophin-mediated apoptosis, for which there is preliminary evidence, and which is suggested to function in vivo as a proof reading mechanism for the elimination of site-inappropriate precursor cells. Aim 3 deals with the mechanisms that direct p75NTR+/TrkC+ cells into the small diameter sensory neuron lineage. Initial data suggest that a subset of neural crest cells differentiates into TrkB-expressing sensory neuroblasts in a chemically defined culture medium in the presence of SCF and absence of any other added growth factors. Using antisense and mutant mouse approaches, it will be determined whether TrkB expression is mediated by SCF and/or by the other autocrine factors, NT-3 and TGF-5. Moreover, BDNF-mediated neuropeptide expression will be documented. A better knowledge of the characteristics of neural crest stem cells has implications for transplant therapy in some degenerative and familial diseases. Moreover, health relatedness pertains to human piebaldism, which is due to c-kit defects. Piebaldism is characterized by hypopigmentation and a white forelock at birth due to faulty melanogenesis. However, the newly detected additional roles of SCF in maintaining stem cells, up-regulating neurotrophin receptors and promoting sensory neurogenesis suggest additional defects in human piebaldism that remain to be elucidated.